Removal of glycosaminoglycans from cultures of human skin fibroblasts

Abstract

Early-passage human skin fibroblasts were grown as monolayers for 2-3 days in minimum essential essential medium containing [35S]sulfate, [3H]glucosamine, [3H]fucose, [3H]proline or [3H]leucine to label proteoglycans, glycoproteins or collagen and other proteins. A crude enzyme preparation obtained from a supernatant from sonicated freeze-dried Flavobacterium heparinum was added to the cell monolayers. This treatment removed most of the 35S-labeled glycosaminoglycans, with no appreciable removal of the 3H-labeled proteins or 3H-labeled glycoproteins. The cells remained attached and viable as a monolayer. The formation of 35S-labeled glycosaminoglycans was examined after pretreating cultures with crude F. heparinum enzyme, followed by addition of fresh growth medium containing [35S]sulfate. The F. heparinum enzyme did not significantly alter the amount or type of 35S-labeled glycosaminoglycans produced. F. heparinum enzyme can be used to provide cultured-cell monolayers depleted of surface glycosaminoglycans. These cells remain attached, viable and subsequently synthesize normal amounts and type of glycosaminoglycans.